PROJECT SUMMARY/ABSTRACT The development of an effective malaria vaccine remains an important global health priority. A benchmark of malaria vaccine efficacy has been the observation that immunization with whole, live Plasmodium sporozoites that are adminstered by mosquito bite, confers complete sterile protection to malaria parasite infection in animal models and controlled human malaria infection (CHMI) trials with Plasmodium falciparum. Recent clinical trials also demonstrated complete protection to CHMI after immunization of human volunteers with parenterally administered attenuated sporozoites. This level of protection has yet to be matched by malaria vaccine candidate subunit formulations. Next to challenges in manufacturing, a formidably challenging aspect of live sporozoite immunization is ensuring safety by means of complete attenuation, while maintaining optimal immunogenicity. There are three main methodologies for live sporozoite immunization: radiation-attenuated sporozoites (RAS), chemoprophylaxis with sporozoites (CPS) and genetically attenuated parasites (GAP). In contrast to CPS and RAS, GAP are attenuated with well- characterized, consistent gene deletions that allow the control of the time point of parasite arrest during liver stage development. This has the potential for greater safety but also for superior efficacy, the latter demonstrated by the finding that late liver stage-arresting rodent malaria GAPs confer long lasting, superior pre-erythrocytic immunity and cross-stage immunity in mice. Furthermore, we have recently engineered a P. falciparum early liver stage-arresting GAP by triple gene deletion that showed for the first time complete pre-erythrocytic attenuation in human volunteers at high dose inoculation by mosquito bite. Here we will build on these successes and propose to design and engineer the next generation of GAP. We will take a two-pronged approach: In Aim 1 we will modify early liver stage-arresting GAP, that show complete attenuation in the P. yoelii rodent malaria model and in P. falciparum, to express blood stage and gametocyte antigens via transgene engineering. In Aim 2 we will generate novel late liver stage-arresting-, fully attenuated GAPs by combinatorial deletion of genes that affect late liver stage development, then augment their cross-stage protective capacity with blood stage and gametocyte antigens via transgene engineering. Finally in Aim 3, we will evaluate the capacity of all novel GAPs to induce completely sterilizing pre-erythrocytic immunity, cross-stage immunity to asexual blood stages as well as sexual stage transmision blocking immunity. We will also elucidate immune mechanisms of protection engendered by immunization with novel GAPs. The ultimate deliverable of this aplication will be a next generation P. falciparum GAP that is safe and is predicted, based on convincing experimental evidence, to afford protection against pre-erythrocytic infection, asexual blood stage parasitemia and will also prevent parasite transmission to the mosquito vector.